초록
<P>The heat treatment of recombinant mesophilic cells having heterologous thermophilic enzymes results in the denaturation of indigenous mesophilic enzymes and the elimination of undesired side reactions; therefore, highly selective whole-cell catalysts comparable to purified enzymes can be readily prepared. However, the thermolysis of host cells leads to the heat-induced leakage of thermophilic enzymes, which are produced as soluble proteins, limiting the exploitation of their excellent stability in repeated and continuous reactions. In this study, <I>Escherichia coli</I> cells having the thermophilic fumarase from <I>Thermus thermophilus</I> (<I>Tt</I>FTA) were treated with glutaraldehyde to prevent the heat-induced leakage of the enzyme, and the resulting cells were used as a whole-cell catalyst in repeated and continuous reactions. Interestingly, although electron microscopic observations revealed that the cellular structure of glutaraldehyde-treated <I>E. coli</I> was not apparently changed by the heat treatment, the membrane permeability of the heated cells to relatively small molecules (up to at least 3 kDa) was significantly improved. By applying the glutaraldehyde-treated <I>E. coli</I> having <I>Tt</I>FTA to a continuous reactor equipped with a cell-separation membrane filter, the enzymatic hydration of fumarate to malate could be operated for more than 600 min with a molar conversion yield of 60% or higher.</P>